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Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro.

Vazquez-Torres A, Jones-Carson J, Mastroeni P, Ischiropoulos H, Fang FC - J. Exp. Med. (2000)

Bottom Line: NO-derived species initially synergize with oxyradicals to kill S. typhimurium, and subsequently exert prolonged oxidase-independent bacteriostatic effects.Interferon gamma appears to augment antibacterial activity predominantly by enhancing NO. production, although a small iNOS-independent effect was also observed.These findings demonstrate that macrophages kill Salmonella in a dynamic process that changes over time and requires the generation of both reactive oxidative and nitrosative species.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.

ABSTRACT
The contribution of the NADPH phagocyte oxidase (phox) and inducible nitric oxide (NO) synthase (iNOS) to the antimicrobial activity of macrophages for Salmonella typhimurium was studied by using peritoneal phagocytes from C57BL/6, congenic gp91phox(-/)-, iNOS(-/)-, and doubly immunodeficient phox(-/)-iNOS(-/)- mice. The respiratory burst and NO radical (NO.) made distinct contributions to the anti-Salmonella activity of macrophages. NADPH oxidase-dependent killing is confined to the first few hours after phagocytosis, whereas iNOS contributes to both early and late phases of antibacterial activity. NO-derived species initially synergize with oxyradicals to kill S. typhimurium, and subsequently exert prolonged oxidase-independent bacteriostatic effects. Biochemical analyses show that early killing of Salmonella by macrophages coincides with an oxidative chemistry characterized by superoxide anion (O(2).(-)), hydrogen peroxide (H(2)O(2)), and peroxynitrite (ONOO(-)) production. However, immunofluorescence microscopy and killing assays using the scavenger uric acid suggest that peroxynitrite is not responsible for macrophage killing of wild-type S. typhimurium. Rapid oxidative bacterial killing is followed by a sustained period of nitrosative chemistry that limits bacterial growth. Interferon gamma appears to augment antibacterial activity predominantly by enhancing NO. production, although a small iNOS-independent effect was also observed. These findings demonstrate that macrophages kill Salmonella in a dynamic process that changes over time and requires the generation of both reactive oxidative and nitrosative species.

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The lucigenin- and luminol-dependent chemiluminescence and NOX production of Salmonella-infected macrophages vary over time. The capacity of IFN-γ–activated macrophages (mφ) to produce ROS and RNS was measured at selected 1-h intervals over a 10-h period from independent wells. The respiratory burst and NOX production were measured as (A) lucigenin- and (B) luminol-dependent chemiluminescence, and (C) by the Griess reaction, respectively. The data are the mean ± SEM of 3–11 independent observations obtained on at least four separate days.
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Figure 4: The lucigenin- and luminol-dependent chemiluminescence and NOX production of Salmonella-infected macrophages vary over time. The capacity of IFN-γ–activated macrophages (mφ) to produce ROS and RNS was measured at selected 1-h intervals over a 10-h period from independent wells. The respiratory burst and NOX production were measured as (A) lucigenin- and (B) luminol-dependent chemiluminescence, and (C) by the Griess reaction, respectively. The data are the mean ± SEM of 3–11 independent observations obtained on at least four separate days.

Mentions: Production of ROS and RNS by IFN-γ–activated macrophages from wild-type, iNOS−/−, and gp91phox−/− mice was determined over a 10-h period by chemiluminescence using lucigenin and luminol, and by spectrophotometry using the Griess reagent (Fig. 4). Production of ROS by macrophages from wild-type mice was initiated immediately after phagocytosis, decreasing to undetectable levels by 6 h thereafter (Fig. 4 A). Lucigenin-dependent chemiluminescence was three times lower than that mediated by luminol (Fig. 4A and Fig. B), suggesting that a substantial proportion of the O2·− formed by the NADPH oxidase reacts with NO· to form ONOO−. As anticipated, macrophages from phox−/− mice exhibited neither lucigenin- nor luminol-dependent chemiluminescence. Macrophages from iNOS−/− mice showed a considerable diminution in luminol-dependent chemiluminescence. However, these macrophages generated a prolonged lucigenin-dependent chemiluminescence that persisted for the duration of the experiment.


Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro.

Vazquez-Torres A, Jones-Carson J, Mastroeni P, Ischiropoulos H, Fang FC - J. Exp. Med. (2000)

The lucigenin- and luminol-dependent chemiluminescence and NOX production of Salmonella-infected macrophages vary over time. The capacity of IFN-γ–activated macrophages (mφ) to produce ROS and RNS was measured at selected 1-h intervals over a 10-h period from independent wells. The respiratory burst and NOX production were measured as (A) lucigenin- and (B) luminol-dependent chemiluminescence, and (C) by the Griess reaction, respectively. The data are the mean ± SEM of 3–11 independent observations obtained on at least four separate days.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193262&req=5

Figure 4: The lucigenin- and luminol-dependent chemiluminescence and NOX production of Salmonella-infected macrophages vary over time. The capacity of IFN-γ–activated macrophages (mφ) to produce ROS and RNS was measured at selected 1-h intervals over a 10-h period from independent wells. The respiratory burst and NOX production were measured as (A) lucigenin- and (B) luminol-dependent chemiluminescence, and (C) by the Griess reaction, respectively. The data are the mean ± SEM of 3–11 independent observations obtained on at least four separate days.
Mentions: Production of ROS and RNS by IFN-γ–activated macrophages from wild-type, iNOS−/−, and gp91phox−/− mice was determined over a 10-h period by chemiluminescence using lucigenin and luminol, and by spectrophotometry using the Griess reagent (Fig. 4). Production of ROS by macrophages from wild-type mice was initiated immediately after phagocytosis, decreasing to undetectable levels by 6 h thereafter (Fig. 4 A). Lucigenin-dependent chemiluminescence was three times lower than that mediated by luminol (Fig. 4A and Fig. B), suggesting that a substantial proportion of the O2·− formed by the NADPH oxidase reacts with NO· to form ONOO−. As anticipated, macrophages from phox−/− mice exhibited neither lucigenin- nor luminol-dependent chemiluminescence. Macrophages from iNOS−/− mice showed a considerable diminution in luminol-dependent chemiluminescence. However, these macrophages generated a prolonged lucigenin-dependent chemiluminescence that persisted for the duration of the experiment.

Bottom Line: NO-derived species initially synergize with oxyradicals to kill S. typhimurium, and subsequently exert prolonged oxidase-independent bacteriostatic effects.Interferon gamma appears to augment antibacterial activity predominantly by enhancing NO. production, although a small iNOS-independent effect was also observed.These findings demonstrate that macrophages kill Salmonella in a dynamic process that changes over time and requires the generation of both reactive oxidative and nitrosative species.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.

ABSTRACT
The contribution of the NADPH phagocyte oxidase (phox) and inducible nitric oxide (NO) synthase (iNOS) to the antimicrobial activity of macrophages for Salmonella typhimurium was studied by using peritoneal phagocytes from C57BL/6, congenic gp91phox(-/)-, iNOS(-/)-, and doubly immunodeficient phox(-/)-iNOS(-/)- mice. The respiratory burst and NO radical (NO.) made distinct contributions to the anti-Salmonella activity of macrophages. NADPH oxidase-dependent killing is confined to the first few hours after phagocytosis, whereas iNOS contributes to both early and late phases of antibacterial activity. NO-derived species initially synergize with oxyradicals to kill S. typhimurium, and subsequently exert prolonged oxidase-independent bacteriostatic effects. Biochemical analyses show that early killing of Salmonella by macrophages coincides with an oxidative chemistry characterized by superoxide anion (O(2).(-)), hydrogen peroxide (H(2)O(2)), and peroxynitrite (ONOO(-)) production. However, immunofluorescence microscopy and killing assays using the scavenger uric acid suggest that peroxynitrite is not responsible for macrophage killing of wild-type S. typhimurium. Rapid oxidative bacterial killing is followed by a sustained period of nitrosative chemistry that limits bacterial growth. Interferon gamma appears to augment antibacterial activity predominantly by enhancing NO. production, although a small iNOS-independent effect was also observed. These findings demonstrate that macrophages kill Salmonella in a dynamic process that changes over time and requires the generation of both reactive oxidative and nitrosative species.

Show MeSH
Related in: MedlinePlus